Nonlinear Chemical Imaging Microscopy: Near-Field Third Harmonic Generation Imaging of Human Red Blood Cells Richard D. Schaller, Justin C. Johnson, and Richard J. Saykally* Department of Chemistry, University of California, Berkeley, California 94720 Third harmonic generation (THG) imaging using a near- field scanning optical microscope (NSOM) is demon- strated for the first time. A femtosecond, tunable near- infrared laser was used to generate both nonresonant and resonantly enhanced third harmonic radiation in human red blood cells. We show that resonantly enhanced THG is a chemically specific bulk probe in NSOM imaging by tuning the excitation source onto and off of resonance with the Soret transition of oxyhemoglobin. Additionally, we provide evidence that tightly focused, nonresonant, far- field THG imaging experiments do not produce contrast that is truly surface specific. There is much current interest in the use of third harmonic generation (THG) as a microscopy tool. 1-5 It is has been shown that third harmonic generation becomes a surface-selective probe when a sample is excited at very high laser intensities and far away from molecular resonances. As a new far-field microscopy contrast mechanism, its utility is quite apparent. Under tight focusing conditions, it appears that the presence of surfaces increases THG efficiency making, for example, transparent cellular organelles and damage spots inside of glass readily observable. 6,7 Such features are difficult to image with more traditional mi- croscopies as they have no visible color and are nonfluorescent. It is the intent of this work to explore the potential of THG as a novel contrast mechanism for near-field imaging. Near-field THG should exhibit characteristics different from those of far-field experiments, primarily due to the relaxation of phase-matching conditions inherent in nonlinear optical near-field scanning optical microscope (NSOM) techniques. 8,9 Conventional wisdom holds that even-order nonlinear optical processes are typically surface specific while odd-order processes are predominantly bulk probes. 10 Second harmonic generation (SHG) can be produced only in materials that lack an inversion center under the dipole approximation, as this condition is explicitly required for  (2) to be nonvanishing. As all interfaces necessarily lack an inversion center, SHG can provide monolayer- sensitive chemical information when the bulk is isotropic. This selection rule involves molecular and translational symmetry elements that are microscopic in nature, as opposed to the phase- matching condition, which is a far-field propagation requirement. Therefore, near-field detected SHG can remain explicitly surface selective when the sample bulk is isotropic. In comparison, THG can be produced from all materials independent of centrosym- metry, because it is dipole allowed. Both surface and bulk molecules will produce third harmonic light, with the bulk typically producing orders of magnitude more signal simply due to the response of more molecules. 10 Within these considerations,  (3) is not intrinsically surface sensitive in the same manner as is SHG. The third-order polarizability induced in a sample is described by the equation 10,12 where  ijkl (3) is a fourth rank tensor that describes the coupling of the three electric fields, E( ω j,k,l ), to create the third harmonic polarization, and i, j, k, and l are electric field orientations with respect to laboratory coordinates.  (3) , in direct analogy to  (2) , consists of both nonresonant and resonant terms where the resonant term can be described by * Corresponding author. E-mail: saykally@ cchem.berkeley.edu. (1) Tsang, T. Y. F. Phys. Rev. A 1995 , 52, 4116-25. (2) Barad, Y.; Eisenberg, H.; Horowitz, M.; Silberberg, Y. Appl. Phys. Lett. 1997 , 70, 922-4. (3) Squier, J. A.; Muller, M.; Brakenhoff, G. J.; Wilson, K. R. Opt. Express 1998 , 3, 315-24. (4) Yelin, D.; Silberberg, Y. Opt. Express 1999 , 5, 169-75. (5) Yelin, D.; Silberberg, Y.; Barad, Y.; Patel, J. S. Appl. Phys. Lett. 1999 , 74, 3107-9. (6) Muller, M.; Squier, J. A.; Wilson, K. R.; Brakenhoff, G. J. J. Microsc. 1998 , 191, 266-74. (7) Squier, J. A.; Muller, M. Appl. Opt. 1999 , 38, 5789-94. (8) Zhao, X.; Kopelman, R. Ultramicroscopy 1995 , 61, 69-80. (9) Vigoureux, J. M.; Girard, C.; Depasse, F. J. Mod. Opt. 1994 , 41, 49-58. (10) Shen, Y. R. Pricnciples of Nonlinear Optics; Wiley: New York, 1984. (11) Schaller, R. D.; Roth, C.; Raulet, D. H.; Saykally, R. J. J. Phys. Chem. B. 2000 , 104, 5217-20. (12) Boyd, R. Nonlinear Optics; Academic: New York, 1992. P i (3) (3ω) R  ijkl (3) E( ω j ) E( ω k ) E( ω l ) (1)  total (3) )  NR (3) +  R (3) (2)  ijkl (3) ( -3ω,ω,ω,ω) ≈ ∑ n,n′,n′′*g <g|i|n> (3ω - ω ng + iΓ ng ) × <n|l|n′′><n′′|k|n′><n′|j|g>+ permutations (2ω - ω n′′g + iΓ n′′g )( ω - ω n′g + iΓ n′g ) (3) Anal. Chem. 2000, 72, 5361-5364 10.1021/ac000699r CCC: $19.00 © 2000 American Chemical Society Analytical Chemistry, Vol. 72, No. 21, November 1, 2000 5361 Published on Web 10/03/2000